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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (5): 700-711.doi: 10.3724/SP.J.1006.2020.94107

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Identification of PEPC genes from foxtail millet and its response to abiotic stress

Jin-Feng ZHAO,Yan-Wei DU,Gao-Hong WANG,Yan-Fang LI,Gen-You ZHAO,Zhen-Hua WANG,Yu-Wen WANG,Ai-Li YU()   

  1. Millet Research Institute, Shanxi Academy of Agricultural Sciences / Shanxi Key Laboratory of Genetic Resources and Breeding in Minor Crops, Changzhi 046011, Shanxi, China
  • Received:2019-07-24 Accepted:2020-01-15 Online:2020-05-12 Published:2020-02-14
  • Contact: Ai-Li YU E-mail:yuailimail@163.com
  • Supported by:
    This study was supported by the Project Plan of Shanxi Academy of Agricultural Sciences(YGJPY2009);This study was supported by the Project Plan of Shanxi Academy of Agricultural Sciences(YGG17021);This study was supported by the Project Plan of Shanxi Academy of Agricultural Sciences(YCX2019T05);the China Agricultural Research System(CARS-06-13.5-A23);the National Agricultural Environmental Data Center Observation and Detection Mission(ZX03S0410)

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Abstract:

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in photosynthesis of C4 plants and plays an important role in a variety of metabolic and stress pathways. In this study, we identified six candidate PEPC genes from foxtail millet genome via sequence alignment. The characteristic parameters of all SiPEPC protein were very similar and the sequences were very conservative. All SiPEPC genes contained the PEPcase motif, which is the characteristic domain of PEPC gene. SiPEPCs were localized in cytoplasm, nucleus and mitochondrion. Promoter analysis identified a variety of light, hormonal, stress, and other growth-related cis-elements in the promoter sequences of SiPEPC members. The qRT-PCR expression profiles showed that the five SiPEPC genes (SiPEPC1, SiPEPC2, SiPEPC3, SiPEPC5, SiPEPC6) were induced by ABA, PEG, high salt and low temperature at seedling stage, indicating that five SiPEPC genes ate involved in abiotic signaling pathway at the seedling stage. The expression of five SiPEPC genes increased with the growth of foxtail millet under normal growth conditions, and increased significantly under drought stress at different growth stages, indicating that five SiPEPCs are involved in drought stress response at jointing, heading and filling stages. The weak light at jointing stage could induce the expression of five SiPEPC genes, while the expression level decreased sharply under moderate light intensity at jointing stage, moderate and weak light intensity at heading and filling stages, showing that light intensity seriously affects the expression of SiPEPC genes.

Key words: foxtail millet, phosphoenolpyruvate carboxylase, abiotic stress, expression analysis

Table 1

Primers used in this study"

基因
Gene		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')
SiPEPC1 TGCGTGCTGGAATGAGTTACT CCAATAAGCATACATCCCGTG
SiPEPC2 CAAGAGCCAAACTATTGACCTG TCATACCAGCACGCATTTCA
SiPEPC3 ATGGAAGGGTGTCCCAAAGTT CCAAGAAGAGAACTGAATGAGAGG
SiPEPC5 GCTACTTCGACGACACCATC AGGAGGAGAACTGGATGAGC
SiPEPC6 GAGGTGGAACTGTGGGAAGA GTAAAACGTTGCAGGGTTCTAA
β-Actin CAGTGGACGCACAACAGGTAT AGCAAGGTCAAGACGGAGAAT

Table 2

Parameters of predicted foxtail millet PEPC genes"

基因
Gene		 基因ID
Gene ID		 染色体位置
Chr. location		 基因长度
Genome
length (bp)		 氨基酸数目
No. of amino acid		 等电点
pI		 分子量
Molecular weight (kD)		 内含子数
Intron number		 不稳定指数
Instability index		 脂肪系数
Aliphatic index		 平均疏水指数
Grand average of hydropathicity		 PEPcse保守域位置
PEPcse domain location
SiPEPC1 Seita.1G020700 1784608-1791259 6652 965 5.83 109.82 9 48.48 87.36 -0.392 163-965
SiPEPC2 Seita.2G173700 26198697-26204510 5814 967 5.77 110.20 9 44.74 92.41 -0.351 164-967
SiPEPC3 Seita.4G175200 28034663-28043265 8603 964 5.95 109.98 8 45.83 85.81 -0.418 162-964
SiPEPC4 Seita.5G074200 6390259-6398514 8256 1032 7.14 114.89 20 52.09 93.87 -0.277 144-348, 414-1032
SiPEPC5 Seita.5G147000 13061164-13065839 4676 1015 5.89 113.53 6 49.05 87.69 -0.319 213-1015
SiPEPC6 Seita.5G324500 37380598-37387338 6741 969 5.70 110.44 9 44.10 89.79 -0.375 165-969

Fig. 1

Gene structure of SiPEPC genes"

Table 3

Prediction of subcellular localization of SiPEPC protein"

基因
Gene		 预测位置
Localization		 可信度
Reliability
SiPEPC1 Cytoplasmic; nuclear; mitochondrial 3.395; 0.685; 0.414
SiPEPC2 Cytoplasmic; nuclear; mitochondrial 3.398; 0.831; 0.317
SiPEPC3 Cytoplasmic; nuclear; mitochondrial 3.679; 0.620; 0.321
SiPEPC4 Cytoplasmic; mitochondrial; nuclear 1.838; 1.290; 1.230
SiPEPC5 Cytoplasmic; mitochondrial; nuclear 2.258; 1.092; 0.472
SiPEPC6 Cytoplasmic; nuclear; mitochondrial 3.428; 0.758; 0.338

Table 4

Putative cis-elements in the promoter of SiPEPCs"

顺式元件
cis-element		 典型序列
Typical sequence		 特性
Characteristic		 基因
Gene
Box 4 ATTAAT Light responsive element SiPEPC1, SiPEPC2, SiPEPC3
G-Box CACGTT Light responsive element SiPEPC1, SiPEPC2, SiPEPC3, SiPEPC4, SiPEPC5, SiPEPC6
GATA-motif AAGGATAAGG Light responsive element SiPEPC1, SiPEPC6
AE-box AGAAACAA Light responsive element SiPEPC2, SiPEPC4, SiPEPC6
GT1-motif GGTTAAT Light responsive element SiPEPC2, SiPEPC4, SiPEPC5
ACE GACACGTATG Light responsive element SiPEPC3, SiPEPC5
Sp1 GGGCGG Light responsive element SiPEPC3, SiPEPC4
ABRE ACGTG Abscisic acid responsiveness SiPEPC1, SiPEPC2, SiPEPC3, SiPEPC4, SiPEPC5
P-box CCTTTTG Gibberellin-responsive SiPEPC1, SiPEPC2
TATC-box TATCCCA Gibberellin-responsiveness SiPEPC2, SiPEPC4
GARE-motif TCTGTTG Gibberellin-responsive element SiPEPC6
TCA-element CCATCTTTTT Salicylic acid responsiveness SiPEPC1, SiPEPC2, SiPEPC5, SiPEPC6
ERE ATTTCATA Ethylene-responsive element SiPEPC2, SiPEPC4
CGTCA-motif CGTCA MeJA-responsiveness SiPEPC3, SiPEPC6
TGACG-motif TGACG MeJA-responsiveness SiPEPC3, SiPEPC6
TGA-element AACGAC Auxin-responsive element SiPEPC1, SiPEPC4
TC-rich repeats GTTTTCTTAC Defense and stress responsiveness SiPEPC1, SiPEPC3
LTR CCGAAA Low-temperature responsiveness SiPEPC2, SiPEPC3, SiPEPC4, SiPEPC6
WUN-motif TTATTACAT Wound-responsive element SiPEPC3
MBS CAACTG MYB binding site involved in drought-inducibility SiPEPC5
ARE AAACCA Anaerobic induction SiPEPC1, 3, 4, 6
CCGTCC-box CCGTCC Meristem specific activation SiPEPC1, 2, 4, 6
GC-motif CCCCCG Anoxic specific inducibility SiPEPC1, 2, 6
O2-site GATGACATGG Zein metabolism regulation SiPEPC2, 4, 5
RY-element CATGCATG Seed-specific regulation SiPEPC3
circadian CAAAGATATC Circadian control SiPEPC5
CAT-box GCCACT Meristem expression SiPEPC6

Fig. 2

Amino acid sequence alignment of PEPCs AtPEPC1, AtPEPC2, AtPEPC3: Arabidopsis thaliana AtPEPC1, AtPEPC2, AtPEPC3; SbPEPC1, SbPEPC2, SbPEPC3: Sorghum bicolor SbPEPC1, SbPEPC2, SbPEPC3; GmPEPC: Glycine max GmPEPC; ZmPEPC1, ZmPEPC2: Zea mays ZmPEPC1, 2; StPEPC: Solanum tuberosum StPEPC; AhPEPC: Arachis hypogaea AhPEPC; RcPEPC: Ricinus communis RcPEPC; NtPEPC: Nicotiana tabacum NtPEPC. The corresponding GenBank protein numbers are Q9MAH0.1, Q5GM68.2, Q84VW9.2, P29195.1, P29194.1, P15804.2, P51061.1, P04711.2, P51059.1, P29196.2, ABY87944.1, ABR29878.1, and P27154.1, respectively. The amino acids with an entire homology are shown by a black background, and those shared non-identical conserved identity by a graybackground (≥ 60% similarity). "

Fig. 3

Phylogenetic relationships of PEPC proteins from different species"

Fig. 4

Expression profile of SiPEPC gene under stresses at seedling stage Light green, dark green, black, light red and dark red are used to represent gene expression levels. Green indicates weak gene expression; red indicates strong gene expression. "

Fig. 5

Relative expression of SiPEPCs during different growth stages under normal and drought conditions * Significantly different at P<0.05; ** Significantly different at P<0.01. "

Fig. 6

Relative expression of SiPEPC under light and drought conditions Light green, dark green, black, light red, and dark red are used to represent gene expression levels. Green indicates weak gene expression; red indicates strong gene expression. J-1: control at jointing stage; J-2: drought at jointing stage; J-3: medium light intensity at jointing stage; J-4: weak light intensity at jointing stage; H-1: control at heading stage; H-2: drought at heading stage; H-3: medium light intensity at heading stage; H-4: weak light intensity at heading stage; F-1: drought at filling stage; F-2: drought at filling stage; F-3: medium light intensity at filling stage; F-4: weak light intensity at filling stage. "

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